Angiogenesis is a multi-step process requiring signals to induce endothelial cell proliferation, migration, and assembly. The Eph receptor tyrosine kinases (RTKs) and their ligands, ephrins, are important mediators of endothelial cell migration and attachment. As membrane tethered ligands, ephrins are capable of transmitting signals bi-directionally. Previous studies from our laboratory have shown that ephrin-B1 is activated by a soluble form of the EphB1-Fc receptor which lacks a cytoplasmic domain and thus is incapable of transmitting forward signals. In addition, soluble EphB1-Fc stimulation of endothelial cells induces cell migration, attachment, and in vivo angiogenesis. These cellular changes require the PDZ binding domain of ephrin B1. Based on these data, we hypothesize that (1) ephrin-B1 reverse signaling is mediated by PDZ adapter proteins through which downstream signaling pathways are activated, and (2) internalization of surface ephrin-B1 represents a mechanism by which ephrin-B1-mediated reverse signaling is regulated. To test these hypotheses, we propose to investigate signaling components downstream of ephrin-B1 by identifying ephrin B1 adapter proteins and pathways that transduce signals affecting endothelial cell migration and adhesion. We will also determine the role of ephrin-B1 internalization in regulating ehrin B1 activation and signal transduction.